The SV40 agnogene product (agnoprotein) was detected in the SV40 lytic infection of African green monkey kidney cells. Antibody to the agnoprotein was raised in rabbits. The agnoprotein was located in the cytoplasm, in the perinuclear region, and on the inner surface of the plasma membrane, but was absent from nucleus by immunofluorescence and subcellular fractionation analysis. A possible regulatory role of this protein in early gene expression is proposed.